Under normal conditions, adaptive behaviors such as eating and sex activate the reward system, and plasticity in the system serves to reinforce and promote these behaviors. Drugs of abuse bypass the normal requirements for stimulation and confer reward directly, altering the properties of the reward system to produce tolerance, physical dependence and drug craving. Drug addiction thus provides a dramatic example of plasticity in the reward pathway. The neurotransmitter dopamine has a crucial role in the reward pathway. Many drugs of abuse increase extracellular dopamine, and repeated intermittent administration can produce larger behavioral responses to subsequent drug exposure, a phenomenon known as behavioral sensitization that has been considered a model for the core features of addiction. Indeed, sensitization to psychostimulants requires the somatodendritic release of dopamine in the ventral tegmental area to induce this form of plasticity, and involves increased dopamine release in the nucleus accumbens in the expression of this plasticity. Together, these observations indicate that dopamine release and its regulation have an important role in the adaptation to drug use. The long-term objective of this proposal is to understand how drugs of abuse alter dopamine release. The strategy is to focus on the membrane trafficking of proteins involved in dopamine release and signal transduction and their regulation. In Project 1, Dr. Sulzer will use amperometry to understand the regulation of dopamine release through a flickering fusion pore, and other biophysical methods to assess the chronic effects of amphetamine on vesicle acidification. He will also embark on a newer line of investigation to study the mechanism and physiological role of structural changes in dopamine release sites. Dr. von Zastrow (Project 2) will examine the mechanisms involved in opioid and dopamine receptor internalization and recycling by real time imaging in primary neuronal culture. Dr. Edwards (Project 3) will investigate the role of regulated exocytosis in the somatodendritic release of dopamine required for behavioral sensitization. In addition, he will explore the relationship between vesicle filling and membrane recycling by studying the functional interaction of VMAT2 with synaptobrevin. In Project 4, Dr. Ryan will characterize the exocytosis and recycling of SVs in dopamine neurons, working with Dr. Sulzer to resolve differences in the nature of release (full fusion versus kiss-and-run), and with Drs. von Zastrow and Edwards on real-time imaging in live neurons, comparing the properties of regulated exocytosis in axon terminals and dendrites.